β-Smac基因的克隆及过表达对胃癌细胞SGC7901顺铂敏感性的影响

Cloning of human β-Smac gene and its dubious effect of enhancing Cisplatin-induced apoptosis on gastric cancer cell line SGC7901

目的:分离,克隆编码β-Smac的基因,观察胃癌细胞SGC7901中β-Smac基因过表达对顺铂敏感性的影响。方法:从胃癌细胞株SGC7901扩增β-Smac的cDNA。构建含β-Smac基因cDNA的表达载体pcDNA3.1-β-Smac。将pcDNA3.1-β-Smac质粒转入SGC7901细胞;用RT-PCR、Western blot分别从核酸和蛋白水平检测外源基因的表达情况。选用顺铂(0、2、8、12μg/ml)处理转染pcDNA3.1(对照组)或pcDNA3.1-β-Smac(实验组)48h后的SGC7901细胞24h,流式细胞仪分析细胞凋亡百分率。结果:成功构建了含β-Smac基因的表达质粒,核酸序列分析显示克隆的β-Smac基因序列与GeneBank中登记的β-Smac基因序列100%同源,引入的Flag序列完全正确。同对照组比较,实验组转染24h后,SGC7901细胞β-Smac mRNA水平明显增高,48h后Western blot在β-Smac预期位置检测到Flag蛋白特异性条带。顺铂处理24h后,仅2μg/ml组显示出实验组和对照组细胞凋亡差异有显著性(P=0.009),而其余各组间均未显示有统计学差异。结论:成功构建含β-Smac基因序列的重组质粒并转染靶细胞,外源性β-Smac基因过表达后,未显示能显著提高人胃癌细胞株SGC7901对顺铂的敏感性。

Objective：Smac is an established pro-apoptosis protein, which moderates the caspase inbitibion of IAPs. This study will clone human β-Smac gene and then investigate whether ectopic overexpression of it would enhance Cisplatin-induced apoptosis on gastric cancer cell line SGC7901. Methods：A pair of PCR primers for β-Smac gene with lower primer containing a sequence of Flag was designed according to the sequence registered in GeneBank.β-Smac fragements amplificated from SGC7901 genome were cloned into plasmid pcDNA3.1 to construct the desired β-Smac transfectants. The discrepancy in β-Smac mRNA expression between cells transfected with β-Smac transfectants or control vectors was investigated by RT-PCR, while the eetopic overexpression of β-Smac protein in cells was detected by western blot. After transfection with either β-Smac transfectants or control vectors for 48h, cells were incubated for another 24 h in the absence or presence of Cisplatin at different concentrations. Then apoptosis was determined by flow cytometry. Results：Nucleotide sequence analysis indicated that the cloned β-Smac sequence was 100% homologous with β-Smac gene registered in GenBank. Compared with cells transfected with control vectors, the β-Smac mRNA expression was significantly increased and ectopie overexpression of β-Smac protein was only detected in cells transfected with β-Smac transfectants. While the difference of apoptotic activity between cells incubated in 2 μg/ml Cisplatin with and without ectopic β-Smac overexpression is statistically significant, there are no statistical differences regarding the other three groups. Conclusion： β-Smac transfectant was successfully constructed and ectopically overexpressed in SGC7901. However, the effec tis conditional and probably three is no effect of ectopic β-Smac overexpression in enhancing Cisplatin-induced apoptosis in SGC7901.